Abstract

The Utah State University iGEM team project is focused on
creating an efficient system for production and monitoring PHA
production in microorganisms. One goal of our research is to develop and
optimize a method, using fluorescent proteins, for the detection of
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in
recombinant E. coli and in Cupriavidus necator. In order to develop an
optimal PHB detection system, we focused on the identification of the
most efficient reporter genes, and the best promoter sequences that
would allow the GFP reporter to indicate maximum PHB production.

Project Objectives

Introduction

Polyhydroxybutyrate

Polyhydroxyalkanoates are a class of over 80 polyesters synthesized by more than 300 microorganisms that exhibit material properties similar to those of petrochemical plastics (Lee, 1999; Lee, 1996). Polyhydroxybutyrate (PHB), the most prevalent PHA, has been of particular interest in commercial and medical applications because of its high tensile strength, biodegradability, and biocompatibility with human tissues (Lee, 1996b; Abe, 1992; Williams 2002). Concerns with the increasingly limited supply of fossil fuels and public anxiety over landfills oversaturated with non-biodegradable materials have also stimulated bioplastic research (Moran, 2006; Ojumu, 2004). PHA research has increased in an effort to understand its potential. It has been used for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005).

PHB Metabolic Pathways

The PHB cassette

Problems with PHB

1. COST

The current cost of industrial PHA production is estimated to be triple that of petrochemical plastics, which limits its widespread use.

2. PRODUCTION EFFICIENCY

Green Fluorescent Protein

GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The Green Fluorescent Protein (GFP) primarily used in this project was GFP E0240 plasmid from the newer iGEM paper.